No

No. followed by 10,000??for 30?min at 4?C. The pellets were resuspended in 100C200?L of sterile 1 phosphate-buffered saline (PBS). Exosomal RNAs were extracted using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA). Plasmids, miR-1246 mimic and inhibitor, and antibodies EJ5-GFP plasmids were linearised by expression levels using a Bio-Rad iCycler and iQ Antxr2 Real-Time PCR system (Bio-Rad) with a fluorescence-labelled SYBR Green Real-Time Master Mix Kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). -actin was used as an endogenous control. The sequences of the forward and reverse primers for these genes and -actin were as follows: ERCC4in the miR-NC group was used to determine the relative expression level Avasimibe (CI-1011) in the treated cells. Cell proliferation assay The cell counting kit-8 (CCK-8) colorimetric assay (DOJINDO Molecular Technologies, Inc., Kumamoto, Japan) was used to assess cell proliferation. To produce the orange coloured product, the WST-8 agent, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt was added to the cell culture medium. The amount of formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. BPE2D cells were transfected with 50?nM of the miR-1246 mimic or miR-NC. After 4?h, the transfected cells were plated in 96-well plates at a denseness of 5103 cells/well and cultured at 37?C in 5% CO2 for the indicated instances. Each sample was assayed in triplicate. Cell viability was identified at 24, 48, and 72?h using the CCK-8 assay. The optical denseness (OD) of each well was read on a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450?nm to determine cell viability. Each experiment was performed in triplicate. Comet and NHEJ restoration effectiveness assay The neutral comet assay, a standard and sensitive technique to analyse DNA DSBs, was used in BEP2D cells.36 BEP2D cells were treated with exosomes following 2?Gy irradiation and transfected with 50 and 100?nM miR-1246 mimic or mimic-NC for 24?h, respectively. Then, cells were trypsinised and resuspended in 1 PBS to a final concentration of 1104 cells/mL. The comet assay was performed using the Comet Assay Reagent Kit for Solitary Cell Gel Electrophoresis (Trevigen, Gaithersburg, MD, USA) according to the manufacturers instructions. Cellular DNA was stained and analysed using an epifluorescence microscope at 40 magnification (Nikon, Melville, NY, USA). The percentage of tail DNA was obtained and quantified using CaspLab software. Additionally, BEP2D cells were transfected with linearised EJ5-GFP, an NHEJ reporter plasmid, and the pmCherry-N1 plasmid. pmCherry-N1 was used like a control to assess transfection effectiveness. After 24?h, the treated BEP2D cells were harvested and analysed using fluorescence-activated cell sorting (FACS) to determine NHEJ restoration effectiveness. Western blot analysis BEP2D cells were lysed in lysis buffer, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Membranes were clogged in 5% milk in Tris-buffered saline comprising Tween-20 (TBST) for 1?h and incubated with the indicated main antibody over night at 4?C. Membranes were then incubated with the indicated secondary antibody for 1?h and washed with TBST. The Image Quant LAS500 system was used to visualise the bands. Details of the western blot Avasimibe (CI-1011) analysis can be found in our earlier study.37,38 Colony-forming ability We performed a colony-forming ability assay to test the effect on BEP2D cell proliferation. Following transfection with the miR-1246 Avasimibe (CI-1011) mimic, inhibitor, or NC, BEP2D cells were seeded onto 60-mm tradition dishes at a denseness of 1000 cells/dish and cultured inside a 5% CO2 incubator at 37?C. After 2 weeks, the cells were stained with crystal violet. The number of microscopic colonies with more than 50 cells was counted. Detection of micronuclei We assessed micronuclei in BEP2D cells.